1 This is the Accepted version of the following article:
2 Melo-Ferreira J, Lemos de Matos A, Areal H, Lissovski A, Carneiro M, Esteves PJ (2015) The
3 phylogeny of pikas (Ochotona) inferred from a multilocus coalescent approach. Molecular
4 Phylogenetics and Evolution 84, 240-244.
5 The original publication can be found here:
6 https://www.sciencedirect.com/science/article/pii/S1055790315000081
7
8 The phylogeny of pikas (Ochotona) inferred from a multilocus coalescent approach
9
10 José Melo-Ferreiraa,*, Ana Lemos de Matosa,b, Helena Areala,b, Andrey A. Lissovskyc, Miguel
11 Carneiroa, Pedro J. Estevesa,d
12
13 aCIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto,
14 InBIO, Laboratório Associado, Campus Agrário de Vairão, 4485-661 Vairão, Portugal
15 bDepartamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4099-002 Porto,
16 Portugal
17 cZoological Museum of Moscow State University, B. Nikitskaya, 6, Moscow 125009, Russia
18 dCITS, Centro de Investigação em Tecnologias da Saúde, IPSN, CESPU, Gandra, Portugal
19
20 *Corresponding author: José Melo-Ferreira. CIBIO, Centro de Investigação em Biodiversidade e
provided by Repositório Aberto da Universidade do Porto
View metadata, citation and similar papers at core.ac.uk CORE brought to you by 21 Recursos Genéticos, Universidade do Porto, InBIO Laboratório Associado, Campus Agrário de
22 Vairão, 4485-661 Vairão. Phone: +351 252660411. E-mail: [email protected].
23
1
1 Abstract
2
3 The clarification of the systematics of pikas (genus Ochotona) has been hindered by largely
4 overlapping morphological characters among species and the lack of a comprehensive molecular
5 phylogeny. Here we estimate the first multilocus phylogeny of the genus to date, by analysing 12
6 nuclear DNA markers (total of 7.5 Kb) in 11 species of pikas from the four classified subgenera
7 (Pika, Ochotona, Lagotona and Conothoa) using a multispecies coalescent-based framework. The
8 species-tree confirmed the subgeneric classification by retrieving as monophyletic the subgenera
9 represented here by more than one species. Contrary to previous phylogenies based on mtDNA
10 alone, Lagotona was found to be sister to Pika. Also, support for the monophyly of the alpina group
11 was not strong, thus caution should be used in future analyses of this group. A relaxed molecular
12 clock calibrated using the Ochotonidae-Leporidae divergence resulted in more recent estimates of
13 divergence times relative to previous studies. Strong concordance with inferences based on fossil
14 records was found, suggesting that the initial diversification of the genus took place by the end of
15 late Miocene. Finally, this work sets up methodologies and gathers molecular markers that can be
16 used to extend the understanding of the evolutionary history of the genus.
17
18 Keywords: Multilocus Coalescent; Ochotona; Pika; Relaxed Molecular Clock; Species-tree;
19 Systematics.
20
21 1. Introduction
22
23 Methods that allow reconstructing the phylogeny of species in a multilocus perspective, taking into
24 account the coalescence of different loci, provide a good opportunity to clarify the systematics of
25 taxonomic groups with traditionally confusing classifications and evolutionary histories. This
2
1 remains true in cases where phylogenies based on the widely used mitochondrial DNA are the sole
2 source of phylogenetic information available at the molecular level, because single-gene
3 phylogenies can often result in erroneous representations of the true species-tree given the variance
4 associated to the evolutionary process among loci (Maddison, 1997). Pikas (family Ochotonidae)
5 are one of such groups.
6
7 Pikas comprise a single extant genus, Ochotona Link, 1795, and with rabbits and hares (family
8 Leporidae) form the order Lagomorpha. Pikas are endemic to the Holarctic Region and the 28
9 recognized living species of pikas are currently mostly restricted to Asia (26 species), with the
10 remaining species inhabiting North America (Lissovsky, 2014). However, pikas are known to have
11 had a more extensive distribution range throughout the Pleistocene. For example, even though the
12 steppe pika (O. pusilla) is today restricted to the central Russian steppes and northern Kazakhstan,
13 fossil records show that during the Pleistocene its range extended to Western Europe (see e.g.
14 Erbajeva and Zheng, 2005 and references therein).
15
16 The attempts to establish a robust phylogeny of pikas have been complicated by the largely
17 overlapping morphological characteristics of extant and fossil species (see Erbajeva and Zheng,
18 2005; Hoffmann and Smith, 2005) and by the use of limited molecular phylogenetic approaches.
19 Phylogenetic relationships in this group were to date inferred solely based on mitochondrial DNA
20 (Yu et al., 2000; Niu et al., 2004; Lanier and Olson, 2009; Ge et al., 2013; Lissovsky, 2014). Some
21 hypotheses have nevertheless resulted from these studies. For example, Yu et al. (2000) suggested
22 that three major evolutionary groups may exist in Ochotona, a northern subgroup, a shrub–steppe
23 dwelling subgroup, and a mountain subgroup, which motivated the partition of species among three
24 subgenera, Pika, Conothoa, and Ochotona, respectively (Hoffmann and Smith, 2005). Later
25 mtDNA phylogenies using a more representative sampling of species suggested some
3
1 rearrangements of taxa among subgenera (Lanier and Olson, 2009; Lissovsky, 2014) or even the
2 inclusion of a fourth subgenus, Lagotona, comprising only O. pusilla (Lissovsky, 2014). However,
3 robust inferences of the relationships among species still await more powerful multilocus analyses.
4
5 Here the first multilocus phylogeny to date – 12 nuclear loci for a total of 7.5 kb – was inferred for
6 11 pika species applying a coalescent-based phylogeny reconstruction method.
7
8 2. Materials and methods
9
10 2.1. Sampling and laboratory work
11
12 A total of 11 pika species, about a third of all presently described Ochotona species, and 12
13 molecular markers were combined in this study. The four subgenera according to Lissovsky (2014),
14 Conothoa, Ochotona, Pika, and Lagotona, were represented in the sampling (Table 1; Fig. 1; see
15 ranges of the sampled species in Smith et al., 1990, Lissovsky et al., 2007 and Smith and Xie,
16 2008), and at least two individuals per species were newly sequenced for each marker. The only
17 exception was O. princeps with one newly sequenced individual to which sequences available in
18 GenBank and Ensembl were added to represent a second specimen (see Suppl. Table 1 for
19 accession numbers). Of the analyzed markers, nine were autosomal (ALB, DARC, OXA1L, PPOX,
20 PRKCI, SPTBN1, TSHB, UCP2 and UCP4; Matthee et al., 2004; Alves et al., 2008; Melo-Ferreira
21 et al., 2009; Melo-Ferreira et al., 2012) and three were X-linked (AMOT, GRIA3 and IL1RAPL1;
22 Carneiro et al., 2010) (see Suppl. Table 2).
23
24 Total genomic DNA was extracted from liver, muscle or testis tissues using the E.Z.N.A. Tissue
25 DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to manufactures’ instructions. The 12
4
1 loci were PCR amplified using the primers indicated in Suppl. Table 2. Sequencing was performed
2 in both directions with an ABI PRISM 3130 Genetic Analyser (Applied Biosystems, Foster City),
3 following the ABI PRISM BigDye Terminator Cycle sequencing protocol.
4
5 2.2. Data Analyses
6
7 The nucleotide sequences were edited using BioEdit (Hall, 1999) and aligned with ClustalW
8 (Thompson et al., 1994). Haplotypes were reconstructed for each individual using PHASE v2.1
9 (Stephens and Donnelly, 2003), implemented in DnaSP v5.10.01 (Librado and Rozas, 2009). The
10 best-fit of several substitution models to each locus was assessed using jModeltest (Posada, 2008)
11 and the Akaike information criterion (AIC). Given that the phylogenetic method to be used (see
12 below) assumes no intra-locus recombination, a second dataset was produced using IMgc (Woerner
13 et al., 2007), retaining the largest non-recombining blocks per locus. A balance between the number
14 of sequences and length was looked for in order to keep at least one sequence per species per locus
15 in the recombination-free data set.
16
17 Sequences from Lepus granatensis were included in the dataset as outgroup. For the X-linked loci,
18 sequences were retrieved from Carneiro et al. (2010) and one additional specimen was newly
19 sequenced for all loci. For the remaining loci, sequences from specimens Lgr2 and Lgr7 from Melo-
20 Ferreira et al. (2012) were used.
21
22 Phylogenetic reconstruction was performed using the multilocus species-tree coalescent-based
23 method implemented in *BEAST v1.8.0 (Drummond et al., 2012) both for the complete and
24 recombination-free alignments. The Yule process and an uncorrelated lognormal relaxed clock
25 model were used. The mutation model was set based on the AIC results of jModeltest, or if the
5
1 specific model was not implemented in *BEAST, the next most parameterized model was selected.
2 Three independent runs of 100 000 000 generations with low autocorrelation of the Markov chain
3 Monte Carlo (MCMC) chain, as examined using Tracer v1.5 (Rambaut and Drummond, 2007),
4 were concatenated using LogCombiner, discarding the first 10% as burn-in. Trees were then
5 summarized with TreeAnnotator, also part of the BEAST package. FigTree v1.3.1
6 (http://tree.bio.ed.ac.uk/software/figtree/) was used to display the inferred species-tree.
7
8 Similarly to the strategy used by Lanier and Olson (2009), calibration of the species-tree was
9 performed considering three possible dates of divergence between Ochotonidae and Leporidae to
10 scale the root mean height: 31 Mya (Matthee et al., 2004), 37 Mya (McKenna and Bell, 1997; Asher
11 et al., 2005) and 65 Mya (Bininda-Emonds et al., 2007). These alternative calibration dates and the
12 95% Highest Posterior Density intervals inferred for node heights, on which no prior constraints
13 were applied, allowed considering a reasonably large range of divergence times, which reflects the
14 uncertainties of molecular dating.
15
16 3. Results and discussion
17
18 3.1. Little influence of recombination on phylogenetic inference
19
20 In this work 11 pika species were analyzed for a total sequence length of 7506 bp (Tables 1 and
21 Suppl. Table 2; GenBank Acc. Nrs. KP292978-KP293227; phased alignments were deposited in
22 Dryad with doi:10.5061/dryad.bn547). Even though this is not the most comprehensive taxonomic
23 sampling of genus performed so far, it represents the first multilocus phylogeny to date, extending
24 the phylogenetic inference performed in other works from one (mtDNA) (Yu et al., 2000; Niu et al.,
25 2004; Lanier and Olson, 2009; Ge et al., 2013; Lissovsky, 2014) to 12 different loci.
6
1
2 A substantial portion of this dataset, three loci (IL1RAPL1, UCP2 and UCP4, for which all
3 sequences of at least one of the species were removed) and 45% of the sequence length of the
4 remaining nine nuclear loci, was lost when retaining only the largest non-recombining blocks,
5 resulting in a total alignment length of 2627 bp (selected mutation models for full and
6 recombination-free datasets are shown in Suppl. Table 3). Removing recombination from the
7 dataset resulted in the same species-tree topology when considering the highly supported nodes
8 (>95% posterior probability), which indicates that recombination had little influence in the
9 phylogenetic reconstruction, in agreement with the conclusions of Lanier and Knowles (2012). We
10 thus opted to present the species-tree based on the full dataset (Fig. 1; but see alternative trees in
11 Suppl. Figs. 1 and 2). The statistically well supported nodes of the species-tree were generally also
12 supported by the individual gene trees sampled in the *BEAST run (Suppl. Table 4).
13
14 3.2. Major evolutionary groups support subgenera classification
15
16 The phylogenetic tree obtained in this work confirms some of the previous inferences based on the
17 analysis of mtDNA alone. The clades represented in Fig. 1 agree with the species arrangement of
18 the Pika, Conothoa and Ochotona subgenera suggested by Lanier and Olson (2009) and Lissovsky
19 (2014). As in these works, the relationships among the three subgenera could not be reliably
20 inferred, and a further increase of the genomic sampling may be needed to solve the polytomy
21 observed at the base of the tree. A major advance was however made in this work associated with
22 the relative placement of O. pusilla in the phylogeny, i.e. of subgenus Lagotona as suggested by
23 Lissovsky (2014). While in previous works this species was either placed at the base of the genus
24 phylogeny (Lanier and Olson, 2009; Lissovsky, 2014), or rather oddly closely related to O.
25 dauurica (subgenus Ochotona) (Ge et al., 2013), our phylogenetic reconstruction strongly suggests
7
1 that Lagotona is sister to Pika. Discordances between gene trees and the species-tree are not
2 surprising and may arise from many different factors, such as incomplete lineage sorting or
3 introgression (which has been shown to particularly affect mtDNA) (Toews and Brelsford, 2012).
4 This and/or the lack of resolution when estimating phylogenies using a relatively short mtDNA
5 fragment may explain the previous difficulties in assessing the evolutionary history of O. pusilla,
6 and highlights the increased power of our multilocus analysis.
7
8 3.3. Molecular dating of diversification and evolutionary relationships among species
9
10 Concordant inferences of node ages between the full and recombination-free datasets (Table 2 and
11 Suppl. Table 5) were obtained. Interestingly, our estimates point to more recent divergence times
12 than those inferred by Lanier and Olson (2009) and Ge et al. (2013), and thus fit better with
13 paleontological estimates (see e.g. Erbajeva and Zheng, 2005), in particular using the most recent
14 calibration point (Ochotonidae-Leporidae divergence of 31 My). McCormack et al. (2011) showed
15 that molecular estimates of divergence dates based on a single locus tend to be overestimated by not
16 taking into account that genetic divergence may predate speciation. This effect can be stronger for
17 mtDNA given its lower effective population size and consequent tendency to more rapidly sort
18 lineages. Our multilocus approach thus seems to provide a more robust estimate of dates of
19 diversification in the genus Ochotona.
20
21 According to fossil records, the formation of the steppe zone in Eurasia which resulted from a
22 global environmental change by the end of late Miocene allowed an explosive radiation of
23 Ochotona (see Erbajeva and Zheng, 2005). Our estimates coincide with this assessment, placing this
24 radiation 6.5-14 Mya (depending on the calibration date used) with the lower bound of the 95%
25 Higher Posterior Density (HPD) at ~4 Mya (node B; Fig. 1; Table 2). In addition, our inference
8
1 suggests that the divergence of Lagotona from subgenus Pika must have occurred shortly after, 4-9
2 Mya (node D; Fig. 1; Table 2).
3
4 Within subgenus Pika, the North American O. princeps was the first to diverge from Asian pikas at
5 Miocene/beginning of Pliocene around 3-7 Mya (node E; Fig. 1; Table 2). Of the remaining six
6 species of Pika analysed here five are morphologically similar (O. hyperborea, O. mantchurica, O.
7 hoffmanni, O. alpina and O. turuchanensis) and form the alpina group. Our phylogeny however did
8 not support the monophyly of this group, because it was not able to resolve the polytomy that
9 includes the alpina group and O. pallasi (node F; Fig. 1). Whether this is due to poor phylogenetic
10 resolution from our 12 loci must be assessed in the future with an increased genetic sampling and
11 larger sample sizes of these species. It nevertheless shows that the monophyly of the alpina group
12 suggested by previous mtDNA-based phylogenies (e.g. Lanier and Olson, 2009; Lissovsky, 2014)
13 should be interpreted with caution. The same occurs at a finer scale. For example, the polytomy at
14 the base of the diversification of O. hyperborea, O. mantchurica and O. hoffmanni (node G; Fig. 1)
15 could not be solved. These are very closely related species that we estimated to have separated 0.8-2
16 Mya, with lower bound of the 95% HPD at 400 kya (Table 2). Likewise, O. alpina and O.
17 turuchanensis were confirmed to be very closely related and to have putatively separated very
18 recently, 278-583 kya (node H, Fig. 1; Table 2), with lower 95% HPD bound at 127 kya. We note
19 that we sampled only a few variants per species and thus cannot confirm the levels of genetic
20 isolation of these taxa. Only a devoted work with a thorough sampling of each of the entities would
21 allow properly capturing the variance of the evolutionary process of divergence (for example in an
22 isolation-with-migration framework; Hey, 2010), shed light onto the degree of genetic isolation and
23 contribute to the discussion of the validity of the specific status of these taxa.
24
25 3.4. Conclusion and future prospects
9
1
2 This work provides a robust view of the evolutionary history of the major groups of the genus
3 Ochotona. Previous molecular phylogenies of the genus were limited to the analysis of a single
4 locus – mtDNA – which could have resulted in misleading inferences. Our results, based on 12
5 independent loci, provide new insights on the phylogenetic position of subgenus Lagotona, sister to
6 Pika, and advise caution when considering the monophyly of the alpina group. This work also made
7 progress in the understanding of the time-frames of the major diversification events within the
8 genus, which tended to be overestimated in mtDNA-based inferences.
9
10 The complete clarification of the phylogeny and systematics of genus Ochotona remains, however,
11 a challenge that should be addressed at both broader and finer evolutionary scales. At a broader
12 scale, increasing the number of sampled species (here we sampled about a third) is fundamental to
13 clarify the composition of the major evolutionary groups and identify the sequence and timing of
14 speciation events in the genus. The methods and molecular markers used in this work appear
15 suitable to address this issue. At a finer scale, larger intraspecific sampling of complexes of closely
16 related species (e.g. the alpina group or the American species) is needed to properly infer the levels
17 of genetic isolation between the forms and contribute with genetic information to the criteria for
18 species classifications. The molecular markers used in this work are also important contributions for
19 such studies. It must however be considered that the recognition of species status should not rely on
20 a simplistic genetic formula and must take into account other sources of information on the biology
21 and history of the organisms in question.
22
23 Acknowledgements
24
10
1 This work was funded by Portuguese National Funds through FCT - Foundation for Science and
2 Technology (FCT-ANR/BIA-BIC/0043/2012 research project), by POPH-QREN funds from the
3 European Social Fund and Portuguese MCTES (FCT, SFRH/BPD/43264/2008 and
4 SFRH/BPD/72343/2010 post-doc grants to JM-F and MC, respectively, and SFRH/BD/48566/2008
5 and SFRH/BD/74948/2010 PhD grants to ALM and HA, respectively), by project “Genomics
6 Applied to Genetic Resources”, co-financed by North Portugal Regional Operational Programme
7 2007/2013 (ON.2–O Novo Norte) under the National Strategic Reference Framework through the
8 European Regional Development Fund, and by the Russian Foundation for Basic Research, grant
9 14-04-00163. Most tissue samples were gently provided by the Zoological Museum of Moscow
10 State University, Russia (Table 1). The DNA sample from American pika was generously provided
11 by the Department of Microbiology and Immunology of Loyola University Chicago, USA. We
12 thank Conrad Matthee and two anonymous reviewers for valuable comments on this manuscript.
13
14 Appendix A. Supplementary material
15
16 Supplementary data associated with this article can be found in the online version.
17
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14
Table 1: Species, geographic location and ID of the specimens newly sequenced in this study
Species Common name Continent Code Sample Locality Specimens id.1 Ochotona alpina Alpine pika Asia Oal1 Republic of Gorno-Altay, Russia S-183539 Oal2 Altayskiy Kray, Russia S-171510
Ochotona dauurica Daurian pika Asia Oda1 Zabaikalskiy Kray, Russia S-182045 Oda2 Zabaikalskiy Kray, Russia S-175916
Oda3 Arahangay Aymak, Mongolia S-183342
Ochotona hoffmanni Hoffmann’s pika Asia Oho1 Zabaikalskiy Kray, Russia S-180456 Oho2 Zabaikalskiy Kray, Russia S-180455
Ochotona hyperborea Northern pika Asia Ohy1 Yakutia Republic, Russia S-183415 Ohy2 Krasnoyarskiy Kray, Russia S-167268
Ochotona mantchurica Manchurian pika Asia Oma1 Zabaikalskiy Kray, Russia S-182046 Oma2 Zabaikalskiy Kray, Russia S-178619
Ochotona pallasi Pallas’s pika Asia Opa1 Bayan-Ulegey Aymak, Mongolia S-183345 Opa2 Republic of Gorno-Altay, Russia S-183540
Ochotona princeps American pika North America Opr1 Unknown - Ochotona pusilla Steppe pika Asia Opu1 Orenburgskaya Oblast'. Russia S-181302 Opu2 Orenburgskaya Oblast', Russia S-181301
Opu3 Orenburgskaya Oblast', Russia S-181303
Ochotona rufescens Afghan pika Asia Orf1 Khorasan, Iran S-178637 Orf2 Khorasan, Iran S-178636
Ochotona rutila Turkestan red pika Asia Ort1 Kashkadarya Region, Uzbekistan S-181326 Ort2 Kashkadarya Region, Uzbekistan S-181325
Ochotona turuchanensis Turuchan pika Asia Otu1 Irkutskaya Oblast', Russia S-171587 Otu2 Krasnoyarskiy Kray, Russia S-162967 1Code from the Zoological Museum, Moscow State University, Moscow, Russia.
15
Table 2: Inferred node ages (in million years) based on three dates of Ochotonidae-Leporidae divergence and the full dataset (see Fig. 1 for node correspondence; 95% Highest Posterior Density intervals in parenthesis).
Calibration (Node A) Node B Node C Node D Node E Node F Node G Node H 31 Mya 6.569 3.015 4.090 3.133 1.462 0.765 0.278 (4.158, 9.157) (1.828, 4.351) (2.546, 5.723) (1.916, 4.469) (0.857, 2.104) (0.442, 1.132) (0.127, 0.443) 37 Mya 7.841 3.599 4.881 3.740 1.745 0.913 0.332 (4.962, 10.929) (2.181, 5.193) (3.038, 6.830) (2.287, 5.334) (1.023, 2.511) (0.527, 1.351) (0.152, 0.529) 65 Mya 13.774 6.322 8.575 6.570 3.066 1.604 0.583 (8.717, 19.200) (3.832, 9.122) (5.338, 11.999) (4.018, 9.370) (1.797, 4.412) (0.926, 2.374) (0.266, 0.930)
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Figure Legend
Fig. 1: Species-tree of Ochotona inferred with *BEAST from the full dataset indicating subgenera according to Lissovsky (2014) and this work. Numbers next to nodes indicate posterior probabilities. Nodes with posterior probability below 95% were collapsed (see full phylogeny in
Suppl. Fig. 1 and recombination-free phylogeny in Suppl. Fig. 2). Inferred mean node ages calibrated with three estimates of Leporidae-Ochotonidae divergence (node A) are shown in Tables
2 and Suppl. Table 5.
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Figure 1:
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